1. Field of the Invention
The present invention relates generally to the field of toxicology and more specifically to the toxicity or side effects to therapeutic and/or other compounds. More particularly, it concerns the development of methods for identifying and screening for substances that can ameliorate the toxic-effects of compounds, such as clinical drugs, food additives, and a variety of other chemical compounds, based upon assaying lymphocytes.
2. Description of Related Art
The impact of chemical intervention on the overall health of specific individuals is often uncertain. While a pharmaceutical may remedy the targeted defect, the treatment may be accompanied by serious side effects that can, in some cases be worse than the initial malady. In general, the side effects of a pharmaceutical preparation are known but may often be limited to a small subset of individuals. In such a subset, side effects may often be so severe that alternate therapies are required. In some cases, the severity of side effects in a small subset of those who would be taking a drug may prevent FDA approval.
In one example, a relatively new class of drugs called xe2x80x98statinsxe2x80x99 function as metabolic inhibitors and are used widely to reduce cholesterol biosynthesis and hence, to reduce levels of cholesterol. The introduction of statins has led to a significant decrease in the mortality and costs associated with ailments involving cholesterol accumulation including atherosclerotic heart disease and stroke. However, a significant number of people who use statins experience side effects. Although the majority of these side effects are mild gasterointestinal ailments several cases of more severe neurological side effects have also been reported. In such cases statin therapy has to be terminated to relieve the side effects. Whether mild or severe side effects in general pose severe deterrents to any drug therapy, especially in the case of drugs that have long-term preventive benefits. Thus, there is an acute need in the art to identify and screen for substances that can counteract side effects of drugs such as statins that cause side effects.
The present inventors have previously developed a specific lymphocyte culture medium and a lymphocyte culture assay which facilitated the assessment of deficiencies or abnormal requirements of nutrients; sensitivity to nutrient imbalances; sensitivity and/or predisposition to side effects of drugs, and/or nutrients, and/or a wide variety of other compounds in an individual (see U.S. Pat. No. 4,499,064, incorporated herein by reference). Although, the inventors previous invention provides methods to predetermine which individuals will experience side effects to a particular compound there is no cost-effective in vitro method in the art to identify substances that can overcome side effects of drugs or other compounds.
The instant invention overcomes the defects in the art and provides a reliable, safe and effective means of identifying substances that can overcome, ameliorate or at least reduce side effects or toxic-effects or sensitivities to different compounds that an individual may experience.
Provided is a method for testing a substance for the ability of the substance to ameliorate the toxic-effects of a compound, comprising the steps of: a) incubating lymphocytes in the presence of the compound and in the presence and absence of the substance; and b) comparing the toxic-effect of the compound on the lymphocytes incubated in the presence of the substance to the effect of the compound on the lymphocytes in the absence of the substance.
In some aspects the method further comprises incubating the lymphocytes in a chemically-defined media. Growing human lymphocytes or any other cell in a chemically-defined medium allows examination of the effects of addition or removal of different components to the medium on cell growth. Chemically-defined media, specifically designed for the growth of human lymphocytes, is described by the present inventors in U.S. Pat. No. 4,499,064, the entire text of which is incorporated herein by reference.
In some embodiments, it is contemplated that the method will further comprise a control comprising: a) incubating the lymphocytes with a compound with a known toxicity in the presence of a substance known to reverse the known toxicity; and b) analyzing the effect on lymphocytes.
In other embodiments, the method further comprises testing a range of concentrations of a substance identified by the method of the invention to obtain the lowest possible dosage at which the substance can reverse the toxic effect of the compound.
In one embodiment, the toxic-effect of the compound is assessed by assessing its effect on the growth of the lymphocytes. In one such embodiment, the growth rate of the lymphocytes is measured. Methods for measuring the growth rate and/or proliferation of lymphocytes are well known in the art. For example, the most widely used method for measurement of cell growth or cell proliferation is assaying the incorporation of tritiated thymidine (3H-thymidine) in cells which is proportional to the rate of DNA synthesis. Other examples include a range of colorimetric procedures such as the MTT assay, the XTT assay, the MTS assay, etc., most of which are based on tetrazolium salts which produce highly colored dyes in response to the metabolic activity of viable cells. The present inventors also intend to use assays that measure the amount and synthesis of ATP in cells using bioluminescence based assays.
In other embodiments, the toxic-effect of the compound is assessed by assessing the effect of such a compound on other responses of lymphocytes. In one such embodiment, the effect of a toxic compound on the cell size of lymphocytes may be measured. In yet another embodiment, a change in the morphology of lymphocytes in response to a toxic compound may be measured. Such measurements may comprise visual identification using microscopic methods. Alternatively, the measurement of a biochemical parameter such as an enzyme activity may be used to access the toxic-effect of the compound on a lymphocyte.
Various types of compounds are known that cause side effects or toxicity. Such compounds include but are not limited to, clinical drugs, teratogenic agents, social drugs, drug additives and stabilizers, food additives, food coloring agents, food components, herbs, herbal extracts, and any other chemical compound that can cause toxicity. The compounds also include agents that are accidentally consumed, for example, certain non-edible mushrooms, non-edible plants, etc.
In one embodiment of the method, the clinical drug is a metabolic inhibitor. In one such aspect the metabolic inhibitor is a statin. The statins are a group of drugs widely used to reduce cholesterol biosynthesis and have proven to be very effective in reducing mortality due to conditions that are caused by cholesterol accumulation in veins such as myocardial infarctions and strokes. Statins are inhibitors of HMG CoA reductase, a liver enzyme involved in cholesterol synthesis. Statins provide a long-term preventive effect on the accumulation of cholesterol. However, a significant percentage of the population suffers from side effects of statins which include a host of mild to severe gasterointestinal effects, muscular inflammation, and peripheral neuropathies. These side effects are remedied by stopping the statin therapy. Given the beneficial effects of statins, termination of statin therapy hinders the long-term health goals sought. The methods of the present invention provide substances that can counteract statin side effects, thereby allowing the continuation of the statin therapy in individuals. In specific aspects, the methods of the invention may be used to identify substances that reverse the toxicity of a statin such as lipitor (Atorvastatin), mevacor (Lovastatin), pravachol (Pravastatin sodium), zocor (Simvastatin), Baychol, Cerivastatin, Fluvastatin and the like.
In another embodiment of the method, the clinical drug is a sedative. Non-limiting examples of sedatives include, but are not limited to, benzodiazepines such as diazepam.
In yet another embodiment, the clinical drug is a chemotherapeutic agent used for cancer therapy and is exemplified by methotrexate, doxorubicin, daunorubicin, mitomycin, actinomycin D, bleomycin, plicomycin, taxol, vincristine, vinblastine, cisplatin, VP16, carmustine, melphalan, cyclophosphamide, chlorambucil, busulfan, lomustine, carboplatin, procarbazine, mechlorethamine, camptothecin, ifosfamide, nitrosurea, tamoxifen, raloxifene, estrogen receptor binding agents, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, or temazolomide.
In still other embodiments of the method, the clinical drug is an anti-inflammatory agent. In specific embodiments, the anti-inflammatory agent may comprise salicylate, and/or acetyl salicylate and may include ibuprofen, ketoprofen, piroxicam, naproxen, naproxen sodium, sulindac, aspirin, choline subsalicylate, diflunisal, oxaprozin, diclofenac sodium delayed release, diclofenac potassium immediate release, etodolac, ketorolac, fenoprofen, flurbiprofen, indomethacin, fenamates, meclofenamate, mefenamic acid, nabumetone, oxicam, piroxicam, salsalate, tolmetin, magnesium salicylate. In other specific embodiments, the anti-inflammatory agent may be a steroid-based agent such as a corticosteroids and is exemplified in non-limiting examples by dexamethason, hydrocortisone, methylprednisolone, prednisone, triamcinolone, etc.
In yet other embodiments of the method, the clinical drug can be isoniazid, valproic acid or retinol.
In one aspect of the invention, the compound can be a teratogenic agent such as thalidomide.
In other aspects of the invention, the compound is a chemical compound such as a food additive, a food component, a color additive, a drug additive and/or a drug stabilizer. Non-limiting examples of food additives include monosodium glutamate, aspartame or saccharin. Non-limiting examples of drug additives include aluminum salts, dyes to color drugs, flavoring agents.
In other embodiments, the compound that causes side effects can be a component of a social drug. For example, various social drugs such as alcoholic beverages comprise ethanol; other social drugs such as coffee, chocolates and some carbonated beverages comprise caffeine; yet other social drugs such as tea""s comprise theophylline, theobromine and caffeine. Side effects caused by such compounds are well known. For example, caffeine, which is a component of coffee, chocolate, colas, and several across the counter drugs, is a central nervous system (CNS) stimulant. At a dosage of 50-100 mg, which is the typical amount in one cup of coffee, an apparent temporary increase in mental clarity and energy levels, with a reduction in drowsiness is seen. At higher doses caffeine causes hyperstimulation. Caffeine also stimulates the cardiovascular system, raising blood pressure and heart rate. Other side effects of caffeine include its diuretic and mild laxative properties. Caffeine and other methylxanthines such as theophylline and theobromine, also increase the respiratory rate and act as bronchodialators. Yet other side effects of higher doses of caffeine intake by pregnant women include birth defects.
The methods of the invention are useful to test substances for their ability to ameliorate toxic-effects and/or side effects of various compounds such as those listed above. However, it will be recognized by the skilled artisan that the invention is in no way limited to the compounds described above and that the methods of the present invention may be used to identify substances that can ameliorate the toxic-effects of virtually any type of chemical or biological compound.
The invention further provides methods that can screen for and identify substances with abilities to reduce toxic-effects. It is contemplated that the substances identified by the method will comprise a wide range of substances such as naturally-occurring substances, and man-made or synthetic substances. These include nutritional supplements, amino acids, proteins, peptides, nucleic acids, oligonucleotides, polynucleotides, lipids, vitamins, drugs, other chemical substances, and other biochemical substances such as those isolated from plants, animals, etc.
The invention also provides a method for ameliorating toxicity in a patient undergoing therapy with a compound that has a known toxicity or side-effect comprising, administrating to the patient a substance identified in accordance with the method of the invention as described above.
It is envisioned that the method of the invention will be useful in the context of human patients. The methods of the present invention may be applicable to other mammalian patients as well. In such embodiments, the media used to culture lymphocytes will be tailor-made to the specific mammal. As will be appreciated by one of skill in the art the nutrient requirements of lymphocytes of different species will vary. Some other mammals contemplated include horses, dogs, cats, cows, sheep.
Also provided by the present invention are methods for ameliorating toxicity in a patient undergoing therapy with a compound that has a known toxicity or side-effect comprising; a) isolating lymphocytes from the patient; b) incubating lymphocytes in the presence of the compound and in the presence and absence of a candidate substance that can reverse the toxicity of the compound; c) comparing the toxic-effect of the compound on the lymphocytes incubated in the presence of the candidate substance to the effect of the compound on the lymphocytes in the absence of the candidate substance thereby identifying a substance that can reverse toxicity; and d) administrating to the patient the identified substance that can reverse toxicity of the compound.
The invention also provides a composition that has the ability to ameliorate the toxic-effects of a metabolic inhibitor. Thus, provided is a composition comprising one or more factors characterized by the following properties: a) isolatable from mammalian plasma; b) containing at least one heat-labile component having a molecular weight of at least 100,000; and c) having the ability to ameliorate the toxicity of a metabolic inhibitor.
In specific aspects, the composition of the invention has the ability to ameliorate the toxic-effects of metabolic inhibitors such as, the statins which are exemplified by, lipitor (Atorvastatin), mevacor (Lovastatin), pravachol (Pravastatin sodium), zocor (Simvastatin), Baychol, Cerivastatin, or Fluvastatin. By xe2x80x98ameliorating the toxic-effectsxe2x80x99 it is meant that the composition can decrease, reduce, eliminate, or reverse the side effects caused by the statins.
The present inventors have also found that the composition comprised of one or more plasma factors is either lacking or deficient in the plasma of certain individuals. The lymphocytes of these individuals show toxic responses to statins. Furthermore, transfer of plasma from an individual whose lymphocytes do not exhibit toxic responses to statins to an individual who lacks or is deficient in the composition reverses or ameliorates the toxic effect.
Thus, in one embodiment, the mammalian plasma from which the composition is can be isolated is human plasma. It is contemplated that one may also use plasma from other mammals such as monkeys, horses, cows, pigs, sheep etc, to isolate the composition.
Additionally, the present invention also contemplates identifying the plasma factors of the composition by testing the efficacy of some known proteins and factors and testing their efficacy at ameliorating statin toxicity. In one such embodiment, the present inventors contemplate testing the LDL-particle for its ability to ameliorate the toxicity of statins. LDL-particles comprise apoB-100 and cholesteryl esters and circulate in the blood stream. LDL receptors on cells recognize these LDL-particles and take up the LDL-particle by endocytosis. The endosome containing the LDL-particle is then translocated to the lysosome where enzymes hydrolyze the cholesteryl esters, releasing cholesterol and fatty acids into the cytosol. The apoB-100 protein is also degraded into amino-acids in the lysosome. As the LDL-particles are involved in the uptake of cholesterol into cells, the present inventors contemplate that they may have a role in the regulation of statin toxicity. As described above and elsewhere in the specification, statins are inhibitors of HMG-CoA reductase, an enzyme involved in the synthesis of cholesterol.
As used herein the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d mean one or more. As used herein in the claim(s), when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d, the words xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d mean one or more than one. As used herein xe2x80x9canotherxe2x80x9d mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.